Molecular biologists have identified the chromosomal defects in a large number of human hereditary diseases, raising the prospects for cures using gene therapy. This emerging branch of medicine aims to correct genetic defects by transferring cloned and functionally active genes into the afflicted cells. Several systems and polymers suitable for the delivery of polynucleotides are known in the art. In addition, gene therapy may be useful to deliver therapeutic genes to treat various acquired and infectious diseases, autoimmune diseases and cancer.
Polycations such as polylysine and DEAE-dextran promote the uptake of proteins and single- and double-stranded polynucleotides into animal cells. Polylysines help assemble DNA into a compact structure, destabilize cell membranes, and provide a handle for the attachment of other effectors to the nucleic acid. The neutralization and condensation of DNA by polylysines into small (ca 100 nm) toroid-like structures, promotes the endocytosis of the nucleic acid into cells in vitro. The endocytic process may be further stimulated by the covalent attachment to the polycation of specific ligands like transferrin, asialoorosomucoid or insulin.
Other useful polynucleotide complexes involve masking the polynucleotide to prevent degradation. Microparticulates, such as erythrocyte ghosts, reconstituted viral envelopes and liposomes have been used in part as protection in gene transfer. One successful liposome system uses the cationic lipid n-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride [DOTMA], mixed with phosphatidylethanolamine [PE] to form the reagent Lipofectin.TM.. Substitutes for DOTMA including lipopolyamine, lipophilic polylysines, and a cationic cholesterol have been used to mediate gene transfer in culture. Other useful lipids include 2,3-dioleyloxy-N-[2-(sperminecarboxyamido)ethyl]-N,N-dimethyl-1-propanamin ium trifluoroacetate [DOSPA], [DOTAP], [DOGG], dimethyldioctadecylammonium bromide [DDAB], cetyldimethylethylammonium bromide [CDAB], cetyltrimethylethylammonium bromide [CTAB], dioctadecyidimethylammonium bromide [DDMB], DL-stearyl stearoyl carnitine ester [DL-SSCE], L-stearyl stearoyl carnitine ester [L-SSCE], DL-stearyl oleoyl carnitine ester [DL-SOCE], DL-palmityl palmitoyl carnitine ester [DL-PPCE], DL-myristyl myristoyl carnitine ester [DL-MMCE], L-myristyl myristoyl carnitine ester [L-MMCE], dioleoylphosphatidylcholine [DOPC], monooleoyl-glycerol [MOG], and cholesterol [Chol]. A difficulty associated with polynucleotide-liposome complexes is that depending on the ratio of the two components, the concentration of the components, and the ionic conditions while mixing, the complexes formed vary greatly in size, in electrostatic properties, in lipid and polynucleotide composition and in ability to interact with biological systems.
Gene transfer efficiency may also be improved by targeting the polynucleotide to the cell of choice. Various procedures based upon receptor mediated endocytosis have recently been described for gene transfer. A cell-specific ligand-polylysine complex was bound to nucleic acids through charge interactions, and the resulting complex was taken up efficiently by the target cells, such as in the case of the human hepatoma cell line HepG2 and of rat hepatocytes in vivo using this delivery system with asialoorosomucoid as a ligand. The stable expression of an enzymatic activity in HepG2 cells following insulin-directed targeting as well as the transferrin-polycation-mediated delivery of a plasmid into the human leukemic cell line K-562 and the subsequent expression of the encoded luciferase gene, have been reported. However, the described delivery systems require the linking of high molecular weight targeting proteins to polynucleotides through a polylysine linker. These large ligand-polycation conjugates also are heterogenous in size and composition, chemically ill-defined, and difficult to prepare in a reproducible fashion.
The use of polycations to neutralize the polynucleotide charge aids the permeabilization of the membrane and the translocation of the polynucleotide. Cationic lipids have also been used for this purpose. Certain cationic lipids termed lipopolyamines and lipointercalants are also known.
Transfection efficiency may also be increased when the polynucleotide is associated with various peptides. Certain useful amphipathic peptides assume .alpha.-helix or .beta.-pleated sheet secondary structures, presenting a charged face and a neutral face. Cationic proteins increase transfection by condensing the polynucleotide.
Other polycations have been shown to promote transfection when associated with polynucleotides. Complexes formed with dendrimers, bulky three-dimensional polymers built by reiterative reaction sequences around a core molecule that may be prepared in varied molecular weights and sizes, efficiently transfer polynucleotides. Other three-dimensional, branched polycations are also useful.
Specific examples of useful self-assembling polynucleotide delivery systems may be found in U.S. patent applications Ser. No. 08/092,200, filed Jul. 14, 1992, and Ser. No. 07/913,669, filed Jul. 14, 1993, which are hereby incorporated in their entirety by reference thereto.
Despite the usefulness of polynucleotide delivery systems, the polynucleotide conjugates can be heterogenous in size and composition, chemically ill-defined, and difficult to prepare in a reproducible fashion. Further, the compound associated with the polynucleotide often is cytotoxic. This greatly limits the concentration at which the complexes can be delivered. Accordingly, there remains a need for means to select desirable polynucleotide complexes and to maximize the permissible concentration of delivered polynucleotide. The present invention satisfies this and other needs.